The broad objective of this proposal is to understand how cells communicate with each other and with the environment in a meaningful way. To find a target surface for adhesion or a final destination for migration, a cell should be able to explore the environment. Filopodia are long slender cellular protrusions which are believed to be the cell's sensory and guiding organelles. They can recognize environmental cues, detect appropriate surfaces and determine the direction of cell locomotion. Filopodia are especially important for navigation of normal tissue cells and for metastasis of cancer cells, but also for initialization of specialized structures, such as synapses, junctions, and communication pathways. This project will focus on studying the structural basis of the dynamic behavior of filopodia during their formation, protrusion, and differentiation into specialized structures. The specific aims are designed to investigate the filopodial machinery in depth with a major focus on interaction of the filopodial cytoskeleton with the plasma membrane, and in breadth, by analyzing the structural and dynamic variations among filopodia formed by different cell types in different conditions and for different purposes. Roles of central players of filopodial machinery, IRSp53 and mDia2, functioning at the interface between the cytoskeleton and plasma membrane will be investigated by combination of structural, kinetic, functional and molecular genetics approaches. The range of filopodia-related structures to be investigated includes leading edge filopodia that control cell motility, dendritic filopodia that establish synapses in brain and differentiate into dendritic spines, and junctional filopodia that are involved in formation of permeability barrier in endothelium. The underlying hypothesis for this project suggests that two molecular machineries, actin cytoskeleton assembly and plasma membrane dynamics, tightly cooperate with each other during filopodia protrusion and this balance can be tuned in different directions to produce a variety of filopodia-based structures serving different functions. A key element of our research strategy is to obtain high resolution structural information by platinum replica electron microscopy and correlate it with the dynamic behavior of the same cell recorded by advanced light microscopy. When additionally combined with modern functional approaches, this strategy has a unique ability to fill the existing gap between properties of individual molecules and behavior of a cell by providing bridging information at subcellular and supramolecular levels. PUBLIC HEALTH RELEVANCE: The results will contribute to understanding of the molecular mechanisms of filopodia formation and the roles of filopodia in such fundamental processes in development and disease as cell migration, cell-cell communication, and tissue morphogenesis. This information will help to design drugs, treatments and diagnostic tools.